Negative-stranded RNA virus (NSV) vectors have shown promise as vaccines against a variety of infectious diseases. While they have similar life-cycles, NSV vectors interact with and modulate innate immunity, such as type I interferon responses, by very different mechanisms. VSV is a highly cytopathic virus that shuts down cellular gene expression by virtue of its M protein. Rabies virus (RV) is a noncytopathic virus that down-modulates STAT function by virtue of its P protein. NDV is an avian paramyxoviruses that appears not to have specific mechanisms of down-modulating innate immunity in mammalian cells. All of these viruses replicate to different extents in vivo and elicit different host signaling programs in dendritic cells. Since it is unclear what constitutes an ideal vector for HIV vaccines, we will now compare these viruses and extract information on how their specific characteristics modulate immunogenicity. A comprehensive analysis of immunogenicity parameters induced by vectors with a similar backbone and replication cycle but with very diverse set of host-virus interactions has not been performed. The overall hypothesis for this application is that NSV vectors can be improved by altering their interactions with the innate immune system. We hypothesize that an effective HIV-1 vaccine must activate dendritic cells (DCs), which are an important link between innate and adaptive immunity. We will first study the impact of these vectors expressing HIV-1 antigens on DC-mediated immunity in-vitro, and identify parameters that would be expected to induce effective adaptive immunity in-vivo, such as efficient antigen presentation and activation. These initial studies will allow us improve the efficiency of our vectors, and provide us with a better basic understanding of what constitutes an optimal vector for the induction of HIV-1-specific immune responses. Once these parameters are determined, we will study the vectors for pathogenicity and immunogenicity in heterologous prime-boost approaches in mice. The most promising approaches identified in mice will be further analyzed in rhesus macaques. PROJECT 1: Examination of modified RV-based HIV-1 vaccines (Schnell, M) PROJECT 1 DESCRIPTION (provided by applicant): Rabies virus (RV) based vaccine vectors induce potent cellular and humoral anti-HIV responses in mice and in rhesus macaques and can provide protection against an HIV-1 like disease in monkeys. However, there is also evidence that such vectors can be improved. Here we perform a detailed study of the activation pattern of RV-infected DCs, which will guide us to construct improved RV vectors. Moreover, we will analyze the impact of type I interferons on DC activation and on CCR7 expression for DC migration. We will analyze the immunogenicity and pathogenicity of such vectors in a mouse model, compare them with Newcastle Disease virus and vesicular stomatitis-based vaccine vectors, and prioritize their use for studies in rhesus macaques. Two specific Aims are proposed: Specific Aim 1. Determine the activation state of DCs after infection with RV-based HIV-1 vaccine vectors and analyze the impact of type I interferon expression by a recombinant RV. Test the hypothesis that interferon type I increases DC activation. Determine the role of CCR7 expression on DC migration. Specific Aim 2 Immunizations of improved NSV HIV-1 vaccine vectors in mice and rhesus macaques. Determine the impact of the innate immune response on the adaptive anti-HIV-1 immune response after infection with optimized RV vectors. Test the hypothesis that activation of innate immune responses reduces vector pathogenicity. Test the hypothesis that two viral vectors induce more potent responses against the expressed HIV-1 antigens than a single vector. Study the efficiency of optimized viral vectors in the rhesus macaque / SIV challenge model system.